Previous work by others has also described the preparation of gold and silver colloids. Such colloids do not have a fixed number of metal atoms and vary considerably in size. For example, the metal colloids can vary in size from 1 nm to 2 .mu.m in size and may contain from about 10 metal atoms to thousands of metal atoms, depending on size. It was found that a number of proteins, such as IgG antibodies, could be adsorbed to these sol particles.
Gold colloids have been most commonly described. These conjugates have been used in electron and light microscopy as well as on immunodot blots for detection of target molecules. These conjugates have many shortcomings. Since the molecules are only adsorbed onto the colloids, they also desorb to varying extents. This leads to free antibody which competes for antigen sites and lowers targeting of gold.
Furthermore, the shelf life of the conjugates is compromised by this problem. The `sticky` colloids also tend to aggregate. If fluorescence is used to detect the target molecules, the gold particles quench most of it. Also, the gold colloids must be stabilized against dramatic aggregation or `flocculation` when salts are added by adsorbing bulky proteins, such as bovine serum albumin. Due to the effects of aggregation and bulky additives, the penetration of immunoprobes into tissues is generally &lt;0.5 .mu.m. Access of the probes to internal cell structures, e.g., nuclear proteins, or to cells deeper in a tissue sample, is impeded by these properties.
Colloidal gold immunoprobes are also used in diagnosis on immunoblots. The sensitivity of these detection schemes is also reduced by problems relating to detachment of antibodies from the gold which results in a short shelf life and non-specific gold binding causing problems with background signal. The gold prepared in standard ways also has low activity due to few adsorbed antibodies and denaturation of some antibodies during adsorption.
Various metal cluster containing organic shells have also been previously described, such as Au.sub.11 (PPh.sub.3).sub.7 Cl.sub.3 (PPh.sub.3 =triphenylphosphine), and Pd.sub.561 L.sub.36 O.sub.200 (where L=1,10-phenanthroline). These metal clusters have a fixed number of metal atoms in their metal cores which range in size from ca. 0.8-2.4 nm. Most of these metal clusters are based upon reduction of metal-triphenyl phosphine or the use of 1,10-phenanthroline.
Examples of larger cluster complexes (greater than lnm in size) have also been reported such as clusters having the formula M.sub.55 (PPh.sub.3).sub.12 X.sub.6 (Ph=phenyl or m-phenylsulfonyl) where M=gold, platinum and rhodium and X=halide.
For example, Barlett, P. A. et al, in "Synthesis of Water-Soluble Undecagold Cluster Compounds . . . ," J. Am. Chem. Soc., 100, 5085 (1978), describe a metal cluster compound (Au.sub.11) having a core of 11 gold atoms with a diameter of 0.8 nm. The metal core of 11 gold atoms in the undecagold metal cluster compound is surrounded by an organic shell of PAr.sub.3 groups. This metal cluster compound has been used to form gold immunoprobes, for example, by conjugating Au.sub.11 to Fab' antibody fragments as well as other biological compounds.
Another metal cluster compound which has been used as a probe is Nanogold.TM. available from the assignee of the present application. Nanogold.TM. has a metal core with 50-70 gold atoms (the exact number not yet being known but believed to be 67 gold atoms) surrounded by a similar shell of organic groups (PAr.sub.3) such that Ar is an aryl group into which a reactive group such as a primary amine, a maleimide, or a N-hydroxysuccinimide ester may be incorporated for conjugation to biologically significant entities including antibody IgG molecules and Fab' fragments, proteins, lipids, hormones and oligonucleotides. Nanogold.TM. and the smaller undecagold cluster, which contains 11 gold atoms, have been used as probes for detecting and identifying biomolecules. The metal core of Nanogold.TM. is 1.4 nm in diameter. The production of Nanogold is described in pending application Ser. No. 988,338, filed Dec. 9, 1992, of James F. Hainfeld and Frederic R. Furuya.
Another class of cluster complex compounds having Pt or Pd as the metal core and further having a core ranging in diameter from 1.8 to 3.6 nm is prepared by reduction of metal acetate in acetic acid by molecular hydrogen in the presence of 1,10-phenanthroline ligands. The ligated cluster is then carefully oxidized with air to neutralize the exposed metal atoms and render the compounds air-stable.
Complexes prepared by the above method and characterized by electron microscopy in order to determine the size of the metal core include a 1.81 nm core diameter platinum compound of proposed formula [Pt.sub.309 phen.sub.35 O.sub.30.+-.10 ] (see Scmidt, G., Morun, B., and Maim, J. -O.; Angew. Chem. Int. Ed. Eng., 1989, 28, 778), a 2.43 nm core diameter palladium compound of proposed formula [Pd.sub.561 phen.sub.36 O.sub.200 ] (de Aguiar, J. A. O.; Brom, H. B.; de Jongh, L. J., and Schmid, G.; Z. Phys. D.: Atoms, Molecules and Clusters, 1989, 12, 457), and a mixture of 3.16 and 3.6 nm core diameter palladium compounds with proposed formulae [Pd.sub.1415 phen.sub.60 O.sub..about.1100 ] and [Pd.sub.2057 phen.sub.84 O.sub..about.1600 ] respectively (Schmid, G.; Harms, M.; Malm, J. -O.; Bovin, J. -O.; van Ruitenbeck, J.; Zandbergen, H. W., and Fu, W. T.; J. Amer. Chem. Soc., 1993, 115, 2046), where phen is either 1,10-phenanthroline or bathophenanthroline, (1). The proposed formulae are based upon the extension of the crystal packing of metal atoms within known smaller clusters outward in discrete layers.
Although the preparation and properties vary for these metal cluster compounds having organic shells, many of these can only be synthesized in low yields, derivatization for use in coupling to biomolecules is expensive in time and effort, and again in low yields, and many of the cluster compounds are degraded rapidly by heat or various chemical reagents.